In gel electrophoresis, a mixture of molecules present in a sample is resolved into its components either partially or completely by differential migration of the components in a gel. The gel can be run positioned vertically or horizontally, and in both formats the samples are usually loaded into sample wells. It is also possible to load the samples directly on a gel surface when that surface is exposed to air, that is, when the gel is run horizontally in a flat-bed mode. Loading the samples directly on the gel surface is not possible in submerged gel electrophoresis, which is known also as submarine electrophoresis, because the sample molecules would spread into the surrounding electrophoresis buffer.
The sample wells are typically formed by means of a comb, which is generally a rectangular piece 5 to 20 cm long, from 0.2 to 3 mm thick and a few centimeters high, and which has numerous protrusions separated by spaces. The comb, also known as sample well-former, is positioned in its place before relation takes place. Following relation the comb is removed, leaving cavities that are of complimentary size and shape. A top view of a gel with 12 sample wells is shown in FIG. 1A. A side view of a similar gel is shown in FIG. 1B. Electrophoresis migration occurs from left to right, as in all other figures below showing similar side views. This design is typical for majority of gels that are currently used for DNA analysis in the submerged gel electrophoresis mode. Prior to loading, a sample solution is mixed with another solution characterized by a high density, commonly known as loading buffer. Since the resulting density of the sample is higher than the density of the running buffer, the sample sinks to the bottom of the well when released from a pipette positioned above the well. In FIG. 1 the loading area is equal to the sample well area, defined by the length and width of the well. If a sample is released anywhere outside that area, it will not enter the sample well.
Modifications of sample wells, and corresponding well-forming devices, are known in the prior art. Thus, U.S. Pat. No. 5,318,682 by Singer describes combs having protrusions of trapezoid shape, which produce sample wells that are wider at the top than at the bottom. This design allows closer spacing of sample wells, and thus makes possible the analysis of a larger number of samples than is possible with rectangular protrusions. Another modification is described in U.S. Pat. No. 4,795,541 by Hard et al. With that modification, a large sample volume can be applied even to very thin gels.
Sample wells of novel design for vertical gels have been disclosed in U.S. Pat. No. 5,304,292 by Jacob et al. Known are also devices that circumvent the use of sample wells. For example, U.S. Pat. No. 5,464,515 by Bellow discloses a device which contains a porous material able to absorb sample molecules prior to loading the samples onto a gel. The absorbed molecules migrate from the porous material to the gel in the electric field. Another way of loading multiple samples simultaneously is disclosed in PCT WO 95/20155 Patent Application by Williams.
The modifications described above have been worked out in order to address specific limitations, or improve certain features, of the manner in which samples are loaded to electrophoresis gels. The ease of sample loading becomes an important issue when many samples have to be processed. Gels with 50 and 100 sample wells are available from Guest Elchrom Scientific under the name Wide Mini S-50 and Wide Mini S-100 gels. These gels were developed for high-throughput applications, and their wells are positioned in 25-well rows. Samples are best applied with a 12-channel pipette, such that alternate wells are filled with one pipetting stroke. Experience has shown that some operators are not always able to deliver all 12 samples precisely to corresponding sample wells without spills into adjacent wells or onto the gel surface. Positioning of 12 pipette tips precisely over 12 alternating wells requires that the operator has steady hands. Even then a problem arises when any one of the 12 pipette tips is not perfectly straight.
Another type of spill is related to PCR samples that have been overlaid with oil. To withdraw a portion of the sample for analysis, the pipette tip needs to pass through an oil layer. Traces of oil then always remain in the sample. Very often this oil does not allow smooth displacement of the sample from the pipette tip into the well. Sometimes the oil causes formation of a "sample bubble" at the pipette tip, which, after bursting, disperses the sample over a wide area.
Yet another source of spills exists during loading of samples onto the gels that are run in submarine mode at a high temperature, for example at 55.degree. C. A part of the sample is often prematurely ejected due to expansion of the air inside the pipette tip after the tip has been placed in the warm running buffer. Regardless of the cause of spills, when molecules from the spilled sample enter the gel, they are subsequently detected as additional bands. Such bands complicate evaluation of the band pattern, and if not recognized, may lead to incorrect interpretation of the experimental results.
One evident way to make sample loading easier is to increase the width and/or length of the wells. However, this approach is associated with serious drawbacks. Thus, the larger the sample wells are, the smaller is the number of samples that can be run on a given gel, making the cost of analysis higher. Moreover, if a multichannel pipette is to be used for sample loading, then sample wells must be spaced according to the spaces which exist between pipette tips. The tips are 9 mm apart in standard multichannel pipettes, so that the distance between the middle of each two adjacent wells must be either 9 mm, for filling each well, or 4.5 mm, for filling alternate wells with one pipetting stroke. Two adjacent wells are usually spaced 1 to 2 mm apart. These dimensions impose a strict limit on the choice of possible lengths of sample wells. On the other hand, increasing the width of sample wells is associated with worsening of resolution. The resolution is related to the width of separated bands, and sharp bands are possible only when molecules enter the gel in a narrow starting zone. In the absence of a stacking gel and a discontinuous buffer system, typical for submerged gel electrophoresis, a narrow starting zone will be achieved only if mobilities of the sample molecules are greatly reduced as they enter the gel. For small molecules, and for low solids content gels, the difference between free mobility and the mobility in a gel is small. Therefore, the width of the starting zone is directly related to the width of the sample well. Consequently, the resolution will be worse in a gel with wide sample wells than in a gel with narrow sample wells, keeping all other parameters constant. In the practice, a compromise is found between band sharpness, the volume that can be loaded into a sample well, and the ease of sample loading.
It has now been found that sample loading can be made easier by a new design of sample wells. With the new design the loading area is enlarged without concomitant increase of the length or width of the sample well. The modification is particularly suitable for gels that are run in the submerged gel electrophoresis mode. The novel design is especially advantageous when loading samples with a multichannel pipette.